ES2356439B1 - SPECIFIC APTÁMEROS FOR HISTONES DE LEISHMANIA. - Google Patents

Abstract

The invention relates to single chain oligonucleotides (aptamers) that are capable of specifically binding to the histone proteins of microorganisms of the Leishmanla genus, as well as to the uses of said oligonucleotides for the diagnosis and treatment of leishmaniasis.

Description

Specific aptamers for histones of Leishmania.

Technical Field of the Invention

The invention relates to single chain oligonucleotides (aptamers) that are capable of specifically binding histone proteins of microorganisms of the Leishmania genus, as well as to the uses of said oligonucleotides for the diagnosis and treatment of leishmaniasis.

Background of the invention

Leishmania is a protozoan that, in the vertebrate, develops an intracellular life cycle, using the macrophage as a host cell. Within these cells, the parasite develops in the phagolysosomes where it is capable of supporting both the acidic pH and the presence of the numerous hydrolytic enzymes that this organelle contains. Protozoa of the genus Leishmania are the causative agent of leishmaniasis, a disease that includes a wide spectrum of clinical symptoms that vary from cutaneous lesions that heal spontaneously to visceral infections that can lead to the death of the patient. Recent WHO data indicate that leishmaniasis is now endemic in 88 countries on five continents - Africa, Asia Europe, North America and South America - with a total of 350 million people at risk of suffering it. It is estimated that this disease affects more than 15,000,000 people worldwide, with its highest incidence in approximately 15 nations, including several countries in the Mediterranean area. As far as America is concerned, it is estimated that 60,000 new cases of American tegumentary leishmaniasis (LTA) occur each year, all located in a region inhabited by some 59 million people. In addition, since 1993, regions in which leishmaniasis is endemic have significantly expanded, which is accompanied by an increase in the number of registered cases of the disease. In addition, AIDS and other immunosuppressive diseases feed the risk of people infected by Leishmania who develop visceral leishmaniasis (WHO 2000).

So far, diagnostic methods for leishmaniasis have been based on the detection of the humoral response to infection in patients. Thus, US patent application US2005287176 describes a chimeric polypeptide formed by several antigenic determinants of different Leishmania proteins arranged in tandem, including antigenic determinants of histone 2A, ribosomal acidic proteins LiP2 and LiP2b, as well as ribosomal protein PO . International patent application WO2005044301 describes the histone 2A sequence and suggests its use for the identification of serum antibodies in patients. European patent application EP0997734 discloses a diagnostic method for the diagnosis of visceral leishmaniasis in which the agglutination of a trystigotide preparation of Leishmania trypsinized and stained with Coomassie bright blue after its addition to a serum sample is investigated.

However, these trials are dependent on the appearance of a humoral response in the patient. Therefore, these types of tests are not useful for the detection of many human leishmaniasis, since these occur primarily in immunosuppressed individuals, who do not develop a complete humoral response. On the other hand, it is common for animals that develop the signs of the disease to appear but in which the antibody titer is not high enough so that the antibodies can be detected.

Therefore, it is necessary to have tests that allow the parasite to be detected instead of tests aimed at detecting the presence of the humoral response in the patient. However, this type of assay faces the problem that there are no commercial antibodies against many of the Leishmania antigens. Patent application EP0819766 describes several Leishmania polynucleotides that can serve as a basis for detection in samples by PCR ampli fi cation using specific primers as well as to distinguish different Leishmania species. Patent application ES216996 describes a method for the detection of L. infantum by PCR ampli fi cation of a specific marker of the kinetoplast followed by ELISA detection of the amplicon generated in the PCR. On the other hand, Moreno, M. et al. (Biochem. Biophys. Res. Commun., 2003, 308: 214218) have described specific DNA aptamers for the KMP-11 protein of L. infantum and have suggested its possible use as a diagnostic tool. However, there is still a need in the art to provide additional methods for the diagnosis of leishmaniasis and, more specifically, of agents capable of binding to Leishmania antigens with sufficient rapidity and affinity to be used for diagnostic purposes.

Compendium of the invention

The present invention is based on the identification by the inventors of Leishmania aptamers that are capable of binding to the histone 2A protein with high affinity, so that they allow the specific detection of said protein without showing any cross-reactivity with the histone 2A of species in which L. infantum usually stays, opening the door to an alternative method for the diagnosis of leishmaniasis.

In a first aspect, the invention relates to a single chain nucleic acid that is capable of specifically binding to a histone of an organism belonging to the genus Leishmania.

In another aspect, the invention relates to an aptamer defined by the formula

5’-GCGGATGAAGACTGGTGT-X-GCCCTAAATACGAGCAAC-3 ’

wherein X is an oligonucleotide selected from the group of SEQ ID NO: 8 to 14 or a functionally equivalent variant thereof or comprising a sequence selected from the group of SEQ ID NO: 1a7 or a functionally equivalent variant thereof.

In another aspect, the invention relates to the use of at least one aptamer of the invention for the detection of Leishmania.

In another aspect, the invention relates to a method of diagnosis of a Leishmania infection comprising

(i)

contacting a biological sample of a patient with at least one aptamer of the invention and

(ii)

detect the union between the aptamer and the sample which is indicative of the presence of Leishmania in the

sample.

In another aspect, the invention relates to an aptamer or a mixture of aptamers of the invention for use in medicine.

In another aspect, the invention relates to a pharmaceutical composition comprising at least one aptamer according to the invention optionally in combination with one or more pharmaceutically acceptable carriers, excipients or solvents.

In another aspect, the invention relates to the use of at least one aptamer of the invention for the preparation of a medicament for the treatment of a Leishmania infection.

In another aspect, the invention relates to a method for the identification of specific aptamers for a histone of an organism of the genus Leishmania comprising

(i)

contacting a histone of a species of the genus Leishmania or a peptide fragment thereof with a library of DNA oligonucleotides,

(ii)

selecting those members of the DNA oligonucleotide library that specifically bind said histone or peptide and

(iii) amplify the oligonucleotides that have been selected in step (ii) to obtain an enriched population of oligonucleotides that bind to the histone or peptide and

(iv) optionally, repeat steps (i) to (iii) one or more times using as a starting material in stage (i) the enriched population obtained in stage (iii).

Brief description of the fi gures

Figure 1 shows the sequence of the 7 clones recovered from screening by SELEX of the oligonucleotide library by affinity against H2A. The graph shows the percentage of each of the nucleotides, pyridines

or pyrimidines in aptamers defined by the sequences SEQ ID NO: 1 to 7. The sequences consist of 76 nucleotides that are formed by the constant regions at positions 5 'and 3', indicated in bold and shown only for the first sequence, and the central variable sequences shown for all aptamers. CG dinucleotides have been underlined.

Figure 2 shows an ELONA titration where the sensitivity of the aptamers of the third round of selection (rd3) and of the LiAPT1 aptamer for the detection of recombinant H2A is compared.

Figure 3 shows the binding kinetics of the aptamer population obtained after the third round of selection and of the LiAPT1 aptamer by ELONA assays for recombinant H2A protein binding.

Figure 4 shows a binding assay of LiAPT1 and LiAPT2 aptamers to increasing amounts of recombinant H2A histone and BSA via slot blot.

Figure 5 shows an immunoblot of total lysates of L. infantum promastigotes (lanes T), cytosolic supernatants (lanes C) or nuclear fractions (lanes N) or the recombinant H2A protein (lanes R). The membranes have been incubated with the LiAPT1 and LiAPT2 aptamers labeled with digoxigenin and visualized with a peroxidase-labeled anti-digoxigenin antibody and a chemiluminescence kit.

Figure 6 shows the results of mapping H2A binding sites to LiAPT1 and LiAPT2 aptamers. A. Sequence of the 9 peptides that cover the entire sequence of L. infantum H2A with an overlap of 5 amino acids at the terminal amino and carboxyl ends. B. ELONA binding studies of peptides # 1 to # 9 and recombinant H2A protein with the population of aptamers obtained after the third round of selection (white bars), with the aptamers LiAPT1 (black bars) and with the aptamer LiAPT2 (white bars). C identification in the three-dimensional structure model of H2A of the position of peptides # 8 and # 5.

Detailed description of the invention

In a first aspect, the invention provides an aptamer comprising a single chain nucleic acid that is capable of specifically binding to a histone of a species of the genus Leishmania.

By aptamer is meant any molecule consisting of a single stranded DNA that is capable of specifically binding to a target substance. By specifier is an aptamer capable of binding to a histone of a species of the genus Leishmania without substantially binding to other proteins of the parasite or to the histones of organisms that act as hosts of Leishmania, including human, canine, bovine or animal histones rodents

The aptamers object of the present can be used for the detection of any Leishmania species, including Leishmania aethiopica, Leishmania amazonensis, Leishmania arabica, Leishmania archibaldo, Leishmania aristedesi, Leishmania (Viannia) braziliensis, Leishmania chagasi (syn. Leishmania infantum), Leishmania ( Viannia) colombiensis, Leishmania deanes, Leishmania donovani, Leishmania enriettii, Leishmania equatorensis, Leishmania forattinii, Leishmania garnhami, Leishmania gerbili, Leishmania (Viannia) guyanensis, Leishmania herreri, Leishmania hertigi, Leishmania leimania, Leishmania lewmania major, Mexican Leishmania, Leishmania (Viannia) naif fi, Leishmania (Viannia) panamensis, Leishmania (Viannia) peruviana, Leishmania (Viannia) pifanoi, Leishmania (Viannia) shawi, Leishmania tarentolae, Leishmania tropic, Leishmania turanica, Leishmania venezia. In a preferred form of the invention, aptamers specifically bind to a histone of L. infantum.

The aptamers object of the present invention have the ability to bind different histones of organisms of the genus Leishmania, in particular, histones H1, H2A, H2B, H3 and H4. In a preferred form of the invention, aptamers specifically bind histones H2A and H3. In an even more preferred form of the invention, aptamers specifically bind histone H2A. The aptamers are capable of specifically binding histones in their native form as well as the variants thereof resulting from their post-translational modification, including the methylated, acetylated, phosphorylated, ubiquitinated, sumolated, citrullinated and ADP-ribosylated variants.

In a preferred embodiment, aptamers specifically bind to the H2A histone of L. infantum.

The aptamers object of the present invention are single chain nucleic acid molecules and include DNA and RNA molecules, as well as variants thereof that have been modified in order to improve the resistance of the molecules to the body's nucleases. Such modifications include modifications in the nucleotides as well as modifications in the phosphodiester bonds.

Modifications in the nucleotides include modifications in the purine or pyrimidine moieties and modifications in the ribose or deoxyribose moieties. Suitable modifications in the context of the present invention include nucleotides modified with arabinose, as described in WO2007 / 038869 as well as nucleotides having in the 2 'position of the sugar a substituent selected from the group of fluorine, hydroxyl, amino, azido, alkyl, alkoxy and alkoxyalkyl. In another embodiment of the invention, the alkyl group is selected from the methyl, ethyl, propyl, butyl group or a functionalized alkyl group such as ethylamino, porpylamino and butylamino. Alternatively, the alkoxy group is methoxy, ethoxy, propoxy or a functionalized alkoxy group according to the formula -Q (CH2) q-R, where is an amino, methoxy or ethoxy group. Suitable alkoxyalkyl groups are methoxyethyl and ethoxyethyl. Aptamers are also part of the invention in which different nucleotides contain different 2 'position modifications.

In another embodiment, the aptamers contain modified bonds such as phosphodiester, phosphotriester, phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoranilothioate, phosphoramidate, methylphosphonate, boranophosphonate as well as combinations of the same or P nucleic acid peptides. , PNA), in which the different nucleotides are linked by amide bonds.

In another embodiment, the aptamers may be conjugated to a molecule in order to modify the pharmacokinetic properties of the aptamer and increase the half-life in the organism. In a preferred form of the invention, aptamers are conjugated to polymers of the carboxymethyl cellulose, chitosan, dextran type

or the polyethylene glycol (PEG), preferably to polymers of the latter having between 10 and 80 kDa in size and, in particular, at least 10, 20, 30, 40, 50, 60, or 80 kDa in size.

In a preferred embodiment, the aptamers of the invention are DNA molecules. In another embodiment, the aptamers of the invention comprise the sequence

5’-GCGGATGAAGACTGGTGT-X-GCCCTAAATACGAGCAAC-3 ’

wherein X is an oligonucleotide selected from the group of SEQ ID NO: 8 to 14 or a functionally equivalent variant thereof. Preferably, the aptamers of the invention comprise a sequence selected from the group of SEQ ID NO: 1a7 or a functionally equivalent variant thereof. Functionally equivalent variant means any aptamer that is derived from the aforementioned oligonucleotides by substitution, addition or deletion of one more nucleotide and that essentially retains the histone binding properties of Leishmania. Methods for establishing whether an aptamer is functionally equivalent to the aptamers of the invention are, for example, the ELONA method as described in example 4, by slot blot as described in example 5, by immunoblotting as and as described in example 7 or by any other method known in the art to detect the binding of one compound to another.

The aptamers object of the invention are capable of specifically binding to the histone H2 of Leishmania without showing substantial cross-reactivity either with other Leishmania proteins or with the histones of the organisms that act as hosts of Leishmania, so that it is possible to use the aptamers of the invention to detect the presence of Leishmania in a sample that is suspected may contain said parasite. Therefore, in another aspect, the invention relates to the use of an aptamer such as defined in claims 1 to 6 or a mixture of said aptamers in a method for the detection of Leishmania.

Leishmania detection can be carried out using any method known in the art to detect the binding of one compound to another. Thus, the use of the aptamers according to the invention can be carried out by ELONA (enzyme linked oligonucleotide assay), as described in US5789163, slot blot, western blot, etc. The use of aptamers for the detection of Leishmania requires the mapping of aptamers with molecules that can subsequently be detected well because they contain directly detectable groups ((for example, fl uorescein, rhodamine, phycoerythrin, coumarin, oxazine, resoruphine, cyanine and derivatives of the same), luminescent groups, QdotTM nanoparticles), through the use of specific compounds for the molecule used as a marker (hapten or antigen / antibodies, biotin / streptavidin, sugar / lecithin, enzyme / cofactor, receptor ligand) or by enzyme mapping which catalyze reactions with fluorogenic, luminogenic or chromogenic substrates. If an enzyme is used, then this enzyme must be able to generate a detectable signal, for example, after the addition of an activator, substrate, amplifying agent and the like. Enzymes that are suitable for the mapping of aptamers according to the present invention and the corresponding substrates include:

•

Alkaline Phosphatase:

◦

Chromogenic substrates: substrates based on p-nitrophenyl phosphate (p-NPP), 5-bromo-4-chloro-3-indolyl phosphate / tetrazolium nitro blue (BCIP / NBT), Fast-Red / Naphthol-AS-TS phosphate

◦

Fluorogenic substrates: 4-methylumbelliferyl phosphate (4-MLJP), 2- (5'-chloro-2'-phosphoryloxyphenyl) -6-chloro4- (3H) -quinazolinone (CPPCQ), 3,6-fl uorescein diphosphate (3 , 6-FDP), diazonic salts of Fast Blue BB, Fast Red TR, or Fast Red Violet LB.

•

Peroxidases:

◦

Chromogenic substrates based on 2,2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), o-phenylenediamine (OPT), 3,3 ', 5,5'-tetramethylbenzidine (TMB), o-dianisidine, acid 5-aminosalicylic, 3-dimethylaxyninobenzoic acid (DMAB) and 3-methyl-2-benzothiazolinehydrazone (MBTH), 3-amino-9-ethylcarbazole (AEC) and 3,3'-diaminobenzidine tetrahydrochloride (DAB).

◦

Fluorogenic substrates: 4-hydroxy-3-methoxyphenylacetic acid, reduced phenoxazines, reduced benzothiazines, including the Amplex® Red reagent, Amplex UltraRed and reduced dihydroxanthenes.

•

Glycosidases:

◦

Chromogenic substrates: o-nitrophenyl-β-D-galactoside (0-NPG), p-nitrophenyl-β-D-galactoside and 4-methylumbelliphenyl-β-D-galactoside (MUG) for β-D-galactosidase.

◦

Fluorogenic substrates: β-D-galactopyranoside fl uorescein, fl uorescein digalactoside (FDG), fl uorescein diglucuronide, 4-methylumbeliphenyl-β-D-galactopyranoside, caroboxiumbeliferyl-β-D-galactopyran uro-galactosopiran io-galactosopiran

•

Oxidoreductases (luciferase):

◦ Luminescent substrates: luciferin.

Alternatively, aptamers can be conjugated to fluorescent nanoparticles to increase detection sensitivity (Smith, J.E. et al. Anal. Chem. 79: 3075-3082).

In another aspect, the invention relates to a method of diagnosis of a Leishmania infection comprising

(i)

contacting a biological sample of a patient with at least one aptamer according to the invention and

(ii)

detect the junction between the aptamer and the sample

where an interaction between the aptamer and the sample is indicative of the presence of Leishmania in the sample.

In principle, any biological sample that contains a certain level of parasitic load can be used in the diagnostic method according to the invention and includes biological fluids (peripheral blood, serum, plasma, urine, synovial fluid, cerebrospinal fluid), scraping or exudate of a skin lesion, spleen or lymph node biopsies.

It is known that aptamers are capable of not only binding to the target molecule but also causing structural alterations in them that may result in loss of activity of the target molecules or may interfere with the interaction of the target protein with other proteins. in the cells (for example, in the case of histone H2A, the aptamers of the invention could block the ability of histone H2A to bind to the other components that form the nucleosome). Therefore, and taking into account the essential role of histones for cell viability, the aptamers of the present invention are also useful as therapeutic agents for the treatment and prevention of all those diseases caused by a Leishmania infection. Thus, in another aspect, the invention relates to the aptamers of the invention or mixtures of various aptamers for use in medicine as well as pharmaceutical compositions comprising at least one aptamer of the invention and, optionally, one or more supports, excipients. or pharmaceutically acceptable solvents.

For use in medicine, the compounds and combinations of compounds of the invention can be formulated together with an excipient that is pharmaceutically acceptable. Preferred excipients for use in the present invention include sugars, starches, celluloses, gums and proteins. In a particular embodiment, the pharmaceutical composition of the invention will be formulated in a pharmaceutical form of solid administration (e.g., tablets, capsules, dragees, granules, suppositories, etc.) or liquid (e.g., solutions, suspensions , emulsions, etc.). In another particular embodiment, the pharmaceutical compositions of the invention can be administered by any route, including, but not limited to, oral, intravenous, intramuscular, intrarterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteric, topical, sublingual or rectal. A review of the various forms of administration of active ingredients, of the excipients to be used and their manufacturing procedures can be found in the Treaty of Farmacia Galenica, C. Faulí i Trillo, Luzán 5, S.A. of Editions, 1993.). Doses and administration regimens can be determined empirically by the expert by widely known methods.

In principle, all types of diseases caused by a Leishmania infection are susceptible to being treated using the method according to the invention. Thus, in a preferred embodiment, the aptamers of the invention are used for the treatment of visceral or kala-azar leishmaniasis, cutaneous leishmaniasis or mucocutaneous leishmaniasis.

The authors of the present invention have identified one of the histone H2A regions of L. infantum that binds to the aptamers LiAPT1 and LiAPT2. Said region extends between amino acids 61 to 80 (the region defined by peptide 5 according to Figure 6). However, similar studies of peptide mapping conducted by Soto et al. (Immunology Letters, 1995, 48: 209-214) demonstrated that isolated sera from leishmaniosis diseased pores recognized almost exclusively the N-terminal and C-terminal regions of the H2A histone of L. infantum, but not the region located between amino acids 61 to 80, which implies that there is a region in histone H2A that is capable of selecting aptamers that specifically bind to it but is unable to act as an immunogen in vivo. The identification of the minimum regions in the H2A histone for the selection and binding of aptamers opens the door for the use of said minimum regions for the identification of aptamers, with the consequent economic advantage since it is not necessary to use the complete protein. Thus, in another aspect, the invention relates to a method for the identification of specific aptamers for a histone of an organism of the genus Leishmania comprising

(i)

contacting a histone of a species of the genus Leishmania or a peptide fragment thereof with a library of DNA oligonucleotides,

(ii)

selecting those members of the DNA oligonucleotide library that specifically bind said histone or peptide and

(iii) amplify the oligonucleotides that have been selected in step (ii) to obtain an enriched population of oligonucleotides that bind to the histone or peptide and

(iv) optionally, repeat steps (i) to (iii) one or more times using as a starting material in stage (i) the enriched population obtained in stage (iii).

The DNA library used in step (i) contains a large number of possible sequences in order to obtain a wide range of binding affinities. Preferably, the number of initial sequences is 1014, but up to 1018 can be used. All the sequences consist of a central region of random sequence, anchored by regions of known sequence so that the oligonucleotides enriched after each stage of binding to the target sequence can be ampli fi ed using primers specific for such regions of known sequence. Preferably, the central region of random sequence consists of 20 to 50 nucleotides and the lateral regions of constant sequence consist of 20 to 40 nucleotides. In a preferred embodiment, the central region consists of 40 nucleotides and each lateral region consists of 28 nucleotides each.

The invention contemplates the use as a target sequence for the selection of oligonucleotides that specifically bind both a histone of the genus Leishmania and a fragment thereof. If the complete histone is used, it may have been produced recombinantly by any of the techniques known to the expert for the production of recombinant proteins, including expression in bacteria (E. coli, B. subtilis), yeasts (S. cerevisae, P. pastoris), insect cells, plant cells or mammalian cells.

In a preferred embodiment of the invention, the target sequence used in step (i) of the method of the invention is an H2A histone peptide comprising a sequence corresponding to amino acids 61 to 80 or amino acids. Acids 106 to 125 of histone H2A from L. infantum. By sequence "corresponding" to the acidic aminos 61 to 80 or 106 to 125 of the H2A histone of L. infantum is understood all that sequence which, in multiple sequence alignment between all histones of the genus Leishmania, overlaps with the region consisting of amino acids 61 to 80 or 106 to 125 of histone H2A of L. infantum. Multiple sequence alignment is obtained using any of the methods known to those skilled in the art, preferably by using the ClustalW algorithm. In another preferred embodiment, the target sequences that are used in the step

(i)

it is a peptide comprising the amino acids 61 to 80 or amino acids 106 to 125 of histone H2A of

L.

infantum

In step (ii), the target proteins or peptides are separated from unbound oligonucleotides. Separation is normally carried out by affinity chromatography with a ligand that is capable of specifically binding to the target protein. In a preferred form of the invention, the target carries an additional sequence that allows its isolation. Virtually any peptide or peptide sequence that allows the isolation or purification of the fusion peptide or protein can be used, for example, a polyhistidine sequence, a peptide sequence recognized by a monoclonal antibody and which can be used to purify the resulting fusion protein by immunoaffinity chromatography, for example, tag peptides such as c-myc, HA, E, FLAG, etc. [Using Antibodies: A laboratory manual. Ed Harlow and David Lañe (1999). Cold Spring Harbor Laboratory Press. New Cork Chapter: Tagging proteins. pp. 347-377] and, in general, any other sequence recognized by an antibody.

In step (iii), the oligonucleotides that have bound to the target sequence are ampli fi ed by a polymerase chain reaction using primers specific for the constant side sequences.

After each round of binding, selection and amplification, the affinity of the oligonucleotide population is increasing while the sequence variability decreases so that, ideally, after several rounds of selection, the final nucleic acid mixture will be composed of a reduced number of different sequences.

The number of selection rounds will depend on the degree of affinity desired or until a significant improvement of the affinity is observed after repeating the selection / amplification cycle. Preferably, the number of binding / selection rounds is from 2 to 20. In a preferred embodiment, the number of selection rounds is from

3.

The invention is described below by examples that are not limiting of the invention, but illustrative.

Example 1

materials

Synthetic random DNA and unlabeled primers were obtained from Metabion (Martinsried, Germany). The primers labeled with digoxigenin were purchased from TIB BIOMOL (Berlin, Germany), bovine serum albumin (BSA) was obtained from Sigma. DMEM and PBS were purchased from GibcoBRL.

Example 2

Expression and puri fi cation of recombinant H2A

The histone H2A of L. infantum cloned into the expression vector pQE30 was purified by Ni-NTA column affinity chromatography as previously described (Iborra, S. et al. 2004, Vaccine, 22: 3865-3876). In summary, the cells expressing H2A were collected and resuspended in 2-5 volumes per gram of wet weight of sonication buffer (8M urea, 0.1M NaH2PO4, 0.01M Tris-HCl pH 8.0). After binding to the Ni-nitroacetic acid column, the recombinant proteins gradually refolded in the column as described (Shi,

P.Y. et al. 1997, Biotechniques, 23: 1036-1038). Subsequently, the recombinant H2A protein was eluted in 0.3M imidazole and dialyzed against PBS at 4 ° C and lyophilized.

Example 3

Selection of aptamers by SELEX

The selection of single stranded DNA aptamers (ssDNA) against Leishmania H2A protein was made from a population of ssDNA oligonucleotides, designated as round 0 (Rd0), which contain a random core region of 40 nucleotides fl apsed by two conserved regions of 28 nucleotides in length each (5'-GCGGATGAAGACTGGTCT-40N-GTTGCTCGTATTTAGGGC-3 '). The ssDNA library is denatured at 90 ° C ice for 10 min. For the initial SELEX selection round (Systematic Evolution of Ligands by Exponential enrichment), 2 nmol of ssDNA were mixed with 2 pg of recombinant H2A protein, which contains a 6 histidine (6xHIS) tail at its amino terminal end, at 200 pl of selection solution (20 mM Tris-HCl pH 7.4, 1 mM MgCl2,150 mM NaCl, 5 mM KCl, 0.2% BSA) and incubated at 37 ° C for 30 min. The aptamer-H2A complexes were purified by adding 20 pL of Ni-NTA resin (Qiagen) for 1 h at 4 ° C. After washing three times with 1 mL of selection solution, the ssDNA-protein complexes were resuspended in 20 pL of distilled water and amplified by PCR using oligonucleotides F3 (5'-GCGGATGAAGACTGGTGT3 ') and R3 (5'- GTTGCTCGTATTTAGGGC-3 ') at a concentration of 1 pM, 250 pM dNTPs, in a final volume of 200 pL at 56 ° C for 25 cycles. The PCR product was precipitated with ethanol. After 3 rounds of amplification selection, the selected aptamer population (SELH2A) was cloned into the pGEM vector and 20 of the clones were sequenced and analyzed.

Example 4

ELONA assay (Enzyme-linked oligonucleotide assay)

Recombinant H2A from L. infantum was diluted to 5 microg / ml (330 nM) in selection buffer and 200 microL of solution (66 pmol per well) was incubated in a 96-well plate overnight at 4 ° C to allow binding to the plate and then washed 4 times in selection buffer. Subsequently, 200 microL of the population of SELH2A aptamers labeled with digoxigenic, of the aptamer LiAPT1 (SEQ ID NO: 1) labeled with digoxigenin or of the population Rd0 also labeled with digoxigenin in selection buffer at different concentrations (0.2 were added to each well -100 nM) that had been previously denatured for 10 min at 95 ° C. The plate was incubated at 37 ° C for 30 min and subsequently, the individual wells were washed 4 times with selection buffer to remove unbound DNA. Subsequently, 200 microL of a 1: 1000 solution of peroxidase-labeled anti-digoxigenin antibody was added to each well. After 30 minutes of incubation, the plates were washed 4 times and developed with an ABTS solution (Boehringer-Mannheim) according to the manufacturer's instructions. The optical density values at 405 nm were determined using a TECAN microplate reader.

In another series of experiments, the recombinant H2A protein was diluted to the appropriate concentrations (1.65-660 nM) in selection buffer and 200 microL of each solution was incubated in a 96-well microtiter plate at 4 ° C overnight. The SELH2A aptamer population labeled with digoxigenin or the LiAPT1 aptamer (SEQ ID NO: 1) also labeled with digoxigenin was diluted in selection buffer to a concentration of 20 mM and denatured at 95 ° C for 10 min and cooled for 10 min. ice. Subsequently, 200 microL of the aptamers were added to each well and the plate was incubated at 37 ° C for 30 min. The individual wells were washed 3 times, the peroxidase-labeled anti-digoxigenin antibody was added and developed using an ABTS solution (Boehringer-Mannheim). Similar experiments were performed by coating the wells with 2 microg of the other Leishmania proteins (H2B, H3, LIP0, LiP2a and LiP2b) to study the speci fi c of aptamers.

Example 5

Slot blot analysis with aptamers

Several quantities were transferred under vacuum of recombinant H2A protein from L. infantum (1.65-33 pmol) and

7.5 pmol of BSA to a nitrocellulose membrane. The filters were raised 3 times in PBS-T for 10 min. And they were blocked with a 5% milk powder solution in PBS-T for 1 h at room temperature. Subsequently, the membranes were incubated with the LiAPT1 and LiAPT2 aptamers labeled with digoxigenin at a concentration of 100 nM in selection buffer with gentle agitation. After the designated time. The membranes were washed with selection buffer 3 times and incubated with a peroxidase labeled anti-digoxigenin antibody diluted 1 to 1000 for 1 hour at room temperature. Excess enzyme was removed by 3 successive washes with selection buffer. Finally, the membranes were revealed with a chemiluminescence kit (Amersham) and exposed to a film. BSA was used as a negative control.

Example 6

Kinetics of binding studies

In order to determine the binding kinetics of the aptamers to H2A, ELONA experiments were performed as described above in which the recombinant H2A protein (66 pmol / well) was incubated with the LiAPT1 aptamer or with the population of SELH2A aptamers labeled in both cases with digoxigenin. Subsequently, the individual wells were washed 3 times, the peroxidase-labeled anti-digoxigenin antibody was added and developed using an ABTS solution (Boehringer-Mannheim).

Example 7

Immunoblotting

Promastigotes of L. infantum were grown in Dulbecco-modified Tagle medium (DMEM) containing 10% fetal serum at 27 ° C. To obtain total protein extracts, promastigotes were sedimented and lysed in lysis buffer (125 mM Tris-HCl at pH 6.8, 4% SDS, 15 mM EXDTA, 20% glycerol, 10% beta-mercaptoethanol and blue of 0.008% bromophenol). Nuclear and cytosolic fractions were separated according to Schreiber et al. (Nucleic Acids Res., 1989, 17: 6419). In summary, 2 x 107 promastigotes were sedimented, washed twice in cold PBS, resuspended in 400 microl of pre-cooled buffer A (10 mM Hepes at pH 7.5, 10 mM KCl, EDTA

0.1 mM, 0.1 mM EGTA, 0.1 mM DTT and 0.5 mM PMSF) and incubated for 15 minutes on ice. After incubation, NP40 (0.6% final) was added and the cells were lysed by vigorous agitation for 10 sec and immediately centrifuged in a microfuge (13,000 g). The supernatant (cytosolic fraction) was mixed in a 1: 1 ratio in 2x Laemmli buffer. The sedimented nuclei were resuspended in 1x Laemmli buffer. Subsequently, the membranes were incubated with the aptamers LiAPT1 (SEQ ID NO: 1) and LiAPT2 (SEQ ID NO: 2) at a concentration of 40 mM selection buffer and incubated for 1 h at room temperature with an anti-digoxigenic antibody labeled with peroxidase diluted 1 to 1000. Subsequently, after 3 washes, the membranes were revealed with a chemiluminescence kit (ECL, Amersham Biosciences) and exposed to a photographic film.

Example 8

H2A-SELH2A interaction mapping

Nine peptides corresponding to the overlapping sequences of the L. infantum H2A protein (10 microg / well) were incubated in a 96-well microanalysis plate overnight at 4 ° C and washed 4 times. Subsequently, the population of SELH2A aptamers labeled with digoxigenin at 0.5 microg / mL were denatured for 10 minutes at 95 ° C, cooled for 10 minutes on ice and 100 microL of the solution was added to each well. Subsequently, the plate was incubated at 37 ° C for 30 minutes, washed four times and peroxidase-labeled antigoxigenin antibodies were added at 37 ° C for 30 min. and revealed using an ABTS solution. Absorbance at 405 nm was determined using a TECAN microplate reader. The peptides were synthesized by the simultaneous multiple solid phase synthetic method using a polyamine resin and FMOC chemistry. The purity was checked by analysis of amino acids and HPLC.

Example 9

Statistic analysis

The results are presented as the mean ± standard error of the mean (SEM) of 3 to 6 independent measures in separate experiments and analyzed using GraphPad Prism v4 (San Diego, CA, USA). The different statistical analyzes are indicated in the text and the figure feet.

Example 10

Selection of anti-H2A aptamers from a combinatorial library

DNA aptamers specific for the L. infantum H2A protein were selected from a single-stranded DNA library formed by a central region of random sequence, anchored at the 5 ’and 3’ ends by constant sequences used for PCR ampli fi cation. The initial library theoretically contained 10141016 different sequences. After mixing with H2A, library oligomers can bind to H2A. During this process, the candidate or the candidates who join can do so specifically to the target and can be recovered with primers F3 and R3. The number of PCR cycles was optimized to avoid excessive amplification. The rigor of the selection was controlled by adjusting the antigen concentrations. Single stranded DNA sequences showing affinity for the target were captured by agarose particles conjugated with nickel. The selected aptamer population (Rd3) was cloned into the pGEM vector and 20 of the clones were sequenced and analyzed, obtaining the sequences shown in Figure 1.

Example 11

Specificity and binding kinetics of anti-H2A aptamers

The H2A binding properties of the final population obtained at the end after three rounds of selection-ampli fi cation (Rd3) and that of the purified LiAPT1 aptamer were compared by the ELONA test. As can be seen in Figure 2, both the enriched population Rd3 and the LiAPT1 aptamer are able to detect the recombinant H2A histone, although the purified aptamer provides a higher signal for the same amount of recombinant protein, indicating that the sensitivity of LiAPT1 for H2A detection is greater than that of Rd3. The binding kinetics of the LiAPT1 aptamer and the Rd3 population were studied by determining the binding of said aptamers to the recombinant protein. The results are shown in Figure 3, where it is observed that in a short time the kinetics of H2A binding to the Rd3 population and the LiAPT1 aptamer are indistinguishable but that in longer times, the LiAPT2 aptamer provides a signal greater than the Rd3 population .

The recognition of the L. infantum H2A protein by the selected clones was carried out by means of slot-blot experiments in which 50 ng and 500 ng of recombinant H2A protein and 500 ng of BSA were transferred to nitrocellulose membranes and proteins. Immobilized were detected with 100 nM of each of the aptamers labeled with digoxigenin. As seen in Figure 4, both aptamers recognize with high affinity the recombinant H2A protein, but showed no affinity for the negative BSA control.

To study the specificity of the selected aptamers, Western blot tests were also carried out in which the total protein and the cytoplasmic and nuclear fractions of promastigotes of L. infantum were immobilized on PVDF membranes and then incubated with the selected aptamers labeled with digoxigenin and developed with anti-digoxigenin-POD antibody. As shown in Figure 5, the selected aptamers specifically recognize endogenous H2A protein both in total proteins and in the nuclear fraction obtained from promastigotes of L. infantum but, however, this band does not appear in the cytoplasmic fraction. In the same experiments, recombinant H2A protein was included as a positive control.

Example 12

Mapping of H2A sites involved in binding to LiAPT1 and LiAPT2 aptamers

To determine the H2A binding sites involved in the interaction with the aptamers of the invention, ELONA assays were carried out with a collection of 9 peptides that completely cover the primary H2A structure (Figure 6A). As shown in Figure 6B, the aptamers LiAPT1 and LiAPT2 recognized peptides 5 and 8 with great specificity (p <0.01; ANOVA followed by Dunnett's test). Likewise, peptide 2 was also recognized although with a markedly lower affinity. Next we decided to locate the peptides in the tertiary structure of H2A. Figure 6C shows that peptides 5 and 8 are positioned forming a pocket region corresponding to one side of the protein. This region is accessible when H2A is part of the nucleosome.

Claims (13)

one.

An aptamer comprising a single chain nucleic acid that is capable of specifically binding to a histone of an organism belonging to the genus Leishmania.

2. An aptamer according to claim 1 wherein the histone to which the aptamer binds is histone H2A.

3.

An aptamer according to claims 1 or 2 in which the organism belonging to the genus Leishmania is Leishmania infantum.

4. An aptamer according to any of claims 1 to 3 wherein the nucleic acid is DNA. 5. An aptamer according to claim 4, wherein aptamer comprises a sequence defined by the following formula: 5’-GCGGATGAAGACTGGTGT-X-GCCCTAAATACGAGCAAC-3 ’ wherein X is an oligonucleotide selected from the group of SEQ ID NO: 8 to 14 or a functionally equivalent variant thereof.

6.

An aptamer according to claim 4, wherein the aptamer comprises a sequence selected from the group of SEQ ID NO: 1a7 or a functionally equivalent variant thereof.

7.

Use of an aptamer according to any one of claims 1 or a mixture of said aptamers in an in vitro method for the detection of Leishmania.

8. Method of diagnosis of a Leishmania infection comprising

(i)

contacting a biological sample of a patient with at least one aptamer such as defined in any of claims 1 to 6

(ii)

detect the union between the aptamer and the sample which is indicative of the presence of Leishmania in the sample.

9.

Method according to claim 8 wherein the Leishmania infection is a visceral, cutaneous or mucocutaneous leishmaniasis.

10.

A pharmaceutical composition comprising at least one aptamer according to any one of claims 1 to 6 optionally in combination with one or more pharmaceutically acceptable carriers, excipients or solvents.

eleven.

Use of at least one aptamer according to any one of claims 1-6 for the preparation of a medicament for the treatment of a Leishmania infection.

12.

Use according to claim 11 wherein the Leishmania infection is a visceral, cutaneous or mucocutaneous leishmaniasis.

SEQUENCE LIST <110> FOUNDATION FOR BIOMEDICAL RESEARCH OF THE RAMÓN Y CAJAL UNIVERSITY HOSPITAL <120> SPECIFIC APTAMEROS FOR HISTONES DE LEISHMANIA <130> P2866ES00 <160> 14 <170> PatentIn version 3.4 <210> 1 <211> 76 <212> DNA <213> Arti fi cial sequence <220> <223> Specific aptamer for the L. infantum H2A antigen (LiAPT1) <220> <221> misc_feature <222> (41) .. (41) <223> nisa, c, g, ort <220> <221> misc_feature <222> (43) .. (43) <223> nisa, c, g, ort <400> 1 <210> 2 <211> 76 <212> DNA <213> Arti fi cial sequence <220> <223> Specific aptamer for L. infantum H2A antigen (LiAPT2) <400> 2 <210> 3 <211> 76 <212> DNA <213> Arti fi cial sequence <220> <223> Specific aptamer for the H2A antigen of L. infantum <220> <221> misc_feature <222> (27) .. (27) <223> nisa, c, g, ort <220> <221> misc_feature <222> (32) .. (32) <223> nisa, c, g, ort <400> 3 <210> 4 <211> 76 <212> DNA <213> Arti fi cial sequence <220> <223> Specific aptamer for the H2A antigen of L. infantum <220> <221> misc_feature <222> (42) .. (42) <223> nisa, c, g, ort <400> 4 <210> 5 <211> 76 <212> DNA <213> Arti fi cial sequence <220> <223> Specific aptamer for the H2A antigen of L. infantum <220> <221> misc_feature <222> (46) .. (46) <223> nisa, c, g, ort <220> <221> misc_feature <222> (51) .. (51) <223> nisa, c, g, ort <400> 5 <210> 6 <211> 76 <212> DNA <213> Arti fi cial sequence <220> <223> Specific aptamer for the H2A antigen of L. infantum <400> 6 <210> 7 <211> 76 <212> DNA <213> Arti fi cial sequence <220> <223> Specific aptamer for the H2A antigen of L. infantum <220> <221> misc_feature <222> (46) .. (46) <223> nisa, c, g, ort <220> <221> misc_feature <222> (50) .. (50) <223> nisa, c, g, ort <400> 7 <210> 8 <211> 40 <212> DNA <213> Arti fi cial sequence <220> <223> Central region of aptamer 1 <220> <221> misc_feature <222> (9) .. (9) <223> nisa, c, g, ort <220> <221> misc_feature <222> (14) .. (14) <223> nisa, c, g, ort <400> 8 <210> 9 <211> 40 <212> DNA <213> Arti fi cial sequence <220> <223> Central region of aptamer 2 <220> <221> misc_feature <222> (23) .. (23) <223> nisa, c, g, ort <220> <221> misc_feature <222> (25) .. (25) <223> nisa, c, g, ort <400> 9 <210> 10 <211> 40 <212> DNA <213> Arti fi cial sequence <220> <223> Central region of aptamer 3 <220> <221> misc_feature <222> (24) .. (24) <223> nisa, c, g, ort <400> 10 <210> 11 <211> 40 <212> DNA <213> Arti fi cial sequence <220> <223> Central region of aptamer 5 <400> 11 <210> 12 <211> 40 <212> DNA <213> Arti fi cial sequence <220> <223> Central region of aptamer 6 <220> <221> misc_feature <222> (28) .. (28) <223> nisa, c, g, ort <220> <221> misc_feature <222> (32) .. (32) <223> nisa, c, g, ort <400> 12 <210> 13 <211> 40 <212> DNA <213> Arti fi cial sequence <220> <223> Central region of aptamer 6 <220> <221> misc_feature <222> (28) .. (28) <223> nisa, c, g, ort <220> <221> misc_feature <222> (32) .. (32) <223> nisa, c, g, ort <400> 13 <210> 14 <211> 40 <212> DNA <213> Arti fi cial sequence <220> <223> Central region of aptamer 7 <400> 14 SPANISH OFFICE OF THE PATENTS AND BRAND Application no .: 200802395 SPAIN Date of submission of the application: 07.08.2008 Priority Date: REPORT ON THE STATE OF THE TECHNIQUE 51 Int. Cl.: See Additional Sheet RELEVANT DOCUMENTS

Category

Documents cited Claims Affected

X

RAMOS E. et al. "A DNA aptamer population specifically detects Leishmania infantum H2A antigen" Laboratory Investigation (2007) vol 87; pages 409-416; DOI10.1038 / labinvest.3700535; whole document. 1-4,7-12

TO

SOTO M. et al. “Mapping of the linear antigenic determinants from the Leishmania infantum histone H2A recognized by sera from dogs with leishmaniasis” Immunology Letters (December 2, 1995) vol. 48; No. 3; pages 209-214; DOI 10.1016 / 0165-2478 (95) 02473-5; whole document. 1-12

TO

SOTO M. et al. “Molecular characterization of a Leishmania donovai infantum antigen identified as histone H2A” European Journal of Biochemistry (April 1992) vol. 205; No. 1; pages 211-216; DOI 10.1111 / j.1432-1033.1992.tb16770.x; whole document. 1-12

Category of the documents cited X: of particular relevance Y: of particular relevance combined with other / s of the same category A: reflects the state of the art O: refers to unwritten disclosure P: published between the priority date and the date of priority submission of the application E: previous document, but published after the date of submission of the application

This report has been prepared • for all claims • for claims no:

Date of realization of the report 21.03.2011

Examiner M. García Coca Page 1/4

REPORT OF THE STATE OF THE TECHNIQUE Application number: 200802395 CLASSIFICATION OBJECT OF THE APPLICATION C12N15 / 115 (2010.01) A61K31 / 711 (2006.01) G01N33 / 569 (2006.01) Minimum documentation sought (classification system followed by classification symbols) C12N, A61K, G01N Electronic databases consulted during the search (name of the database and, if possible, terms of search used) INVENTIONS, EPODOC State of the Art Report Page 2/4  WRITTEN OPINION Application number: 200802395 Date of Written Opinion: 21.03.2011 Statement

Novelty (Art. 6.1 LP 11/1986)

Claims Claims 5, 6 1-4, 7-12 IF NOT

Inventive activity (Art. 8.1 LP11 / 1986)

Claims Claims 5, 6 1-4, 7-12 IF NOT

The application is considered to comply with the industrial application requirement. This requirement was evaluated during the formal and technical examination phase of the application (Article 31.2 Law 11/1986).  Opinion Base.- This opinion has been made on the basis of the patent application as published. State of the Art Report Page 3/4  WRITTEN OPINION Application number: 200802395 1. Documents considered.- The documents belonging to the state of the art taken into consideration for the realization of this opinion are listed below.

Document

Publication or Identification Number publication date

D01

RAMOS E. et al. 2007

2. Statement motivated according to articles 29.6 and 29.7 of the Regulations for the execution of Law 11/1986, of March 20, on Patents on novelty and inventive activity; quotes and explanations in support of this statement The object of the invention, as set forth in claims 1-12, is an aptamer (single stranded DNA) capable of specifically binding to the H2A histone of Leishmania infantum (reiv. 1-6). The use of said aptamer for the detection of Leishmania (reiv. 7) and for the preparation of a medicament for the treatment of leishmaniasis (reiv. 11 and 12), the method of diagnosis of a Leishmania infection is also an object of the invention. (reiv. 8 and 9) and a pharmaceutical composition comprising said aptamer (reiv. 10).  Novelty and Inventive Activity Document D01 discloses single stranded DNA aptamers that specifically bind to the H2A histone of Leishmania infantum. Said aptamers are formed by a variable sequence of 40 nucleotides flanked by two conserved sequences of 18 nucleotides each. This document shows the specificity of these aptamers, recognizing the H2A histone of L. Infantum making them excellent candidates both for the diagnosis and for the therapy of L. Infantum infection, since it does not recognize histones of other species ( for example the human ones), or other proteins of the same species. The object of the invention set forth in claims 1-4 and 7-12 derives directly and without any ambiguity from document D01. Therefore, the invention as set forth in said claims is novel and does not imply inventive activity (art. 6.1 and art. 8.1 Patent Law 11/1986). State of the Art Report Page 4/4